Despite 30 years of AIDS research, there is still no robust, reverse-genetic system for studying the in vivo function of human genes that regulate HIV-1 replication, pathogenesis, and immunity. As the number of human genes suspected of influencing HIV-1 skyrockets, the need for such technology has never been greater. This proposal will address technical hurdles that must be overcome before such an experimental system can be realized. RNAi revolutionized how molecular biologists study human gene function but lack of reproducibility and the inability to assess allelic variants are some of the limitations with this methodology. Mouse gene knockout technology is superb at unambiguously assigning function to particular mammalian genes. Unfortunately, many human genes are not shared with the mouse, including APOBEC3G and TRIM5alpha, two genes that potently restrict HIV-1 replication in tissue culture. Additionally, HIV-1 does not replicate in mouse cells. The project proposed here will develop tools for targeted gene replacement by homologous recombination in cells of the human immune system and for the functional assessment of these modified cells within the context of an in vivo model for HIV-1 transmission, replication, immunity, and AIDS pathogenesis. Towards this end, we will exploit technical developments from the past ten years, including derivation of human embryonic stem cells, reprogramming of human somatic cells into induced pluripotent stem cells, improved reconstitution of immunodeficient mice with human hematopoietic stem cells, development of lentiviral vectors that permit efficient delivery of DNA to transfection-resistant cells, and designer nucleases to stimulate homologous recombination. Development of a perpetual source of isogenic hematopoietic stem cells that can be genetically modified, along with improvements in humanized mouse models, will permit us to draw firm conclusions concerning the function of particular human genes - or of particular alleles - in hematopoietic development, immune function, and in HIV-1 replication and pathogenesis.